Steady and promising advances have been made over the last two decades in chimeric antigen receptor (CAR)-mediated T cell (T-body) therapy. These efforts have led to major breakthroughs in the treatment of B cell malignancies and neuroblastomas in recent clinical trials. However, broader and more rapid implementation of this promising immunotherapy modality has been hindered by the labor-intensive, time-consuming, and expensive ex vivo T cell modification and expansion procedures. Furthermore, monitoring of therapeutic efficacy during the course of treatment remains suboptimal, relying on peripheral blood sampling methods that provide only a snapshot of therapeutic efficacy. To address these logistical and therapy management limitations, we are proposing to develop a combined gene delivery/imaging system based on targeting lentivectors that can specifically transduce T cells in situ with tumor-antigen specific CAR genes and herpes simplex virus-1 thymidine kinase (HSV1tk), a positron emission tomography reporter (PET) gene. This direct gene delivery approach will eliminate the need to genetically modify and expand T cells ex vivo and will provide a means of noninvasively monitoring the biodistribution, trafficking, and targeting of T-bodies to tumor tissue by PET using the nucleoside analog substrate 2'-deoxy-2'-[18F]fluoro-5-ethyl-1-?-D-arabinofuranosyl- uracil ([18F]-FEAU). Expression of HSV1tk will also enable selective ablation of transduced cells in the event of any adverse immune response. Our unique multifunctional gene delivery/imaging system is based on the central hypothesis that direct and specific in situ delivery of tumor antigen-specific CAR and HSV1tk to endogenous T cells by lentiviral vectors (via CD3-binding receptors) will redirect their specificity toward tumo cells and enable noninvasive and repetitive imaging of their targeting patterns by PET. This subset of T-bodies will further expand upon contact with target antigen. Our long-term goal is to develop an off-the-shelf lentiviral system that can be customized to express any tumor antigen-specific CAR and a PET reporter/suicide gene to inject into patients to redirect their T cells toward tumor cells while monitoring therapeutic progress by PET. As a step toward achieving our long-term goal, we are pursuing this multi-PI R01 funding mechanism to develop a prototype lentivector that will deliver erbB-2-specific CAR and HSV1tk to endogenous T cells in a mouse breast cancer model. A diverse team of investigators, headed by Professor Zelig Eshhar (PI) who pioneered CAR-based immunotherapy, will contribute expertise in the fields of CAR development, T cell targeting, and PET imaging to develop this multifunctional gene delivery/imaging therapeutic modality. Successful completion of the proposed work will have a significant impact on the implementation of cancer immunotherapy providing a larger number of patients access to promising CAR-based immunotherapy trials and expanding the application of immunotherapy to a host of malignancies.